The present invention relates to analytical chemistry and, more particularly, to devices and methods which provide for selectively binding chemical species to a substrate. A major objective of the present invention is to provide for more convenient and effective chemical binding to a substrate used in the context of a mass biosensor.
Preservation of the environment requires that the amounts of various pollutants on land and in water be monitored. Laboratories monitoring these pollutants are charged with measuring microquantities of many different chemicals. Mass biosensors provide a valuable tool in this application, as well as in medical and other applications.
Mass biosensors are used to measure microquantities of biological components and have the potential for detecting trace amounts of biological and chemical components. One type of mass biosensor uses a piezoelectric crystal as an acoustic waveguide. An input transducer generates a periodic acoustic wave from a periodic electrical input signal. The acoustic wave propagates through the crystal to an output transducer which converts the received acoustic wave to an electrical output signal. The acoustic wave undergoes a change in propagation velocity which corresponds to the mass bound to the surface of the crystal. By monitoring the frequency or relative phases of the input and output electrical signals, the mass changes at the surface of the crystal can be measured.
To measure the amount of a specific chemical component in a sample solution, the surface of the crystal must be prepared to bind that component selectively. In one approach, a scientist obtains an unmodified crystal and prepares it shortly before component measurement so that it acquires an affinity for the component of interest. For example, an antibody can be bound to a crystal surface to prepare the mass biosensor to measure the amount of the corresponding antigen.
Heretofore, piezoelectric crystal biosensors were constructed so that antibody proteins which bind antigens or antibody-binding proteins which bind antibodies were bound directly to the surface. A major drawback of this method is the extensive amount of time necessary to bind these proteins to the surface, a procedure taking many hours. In some cases, the preparation procedures take 24 hours or more.
Another drawback is that the shelf life of the sensors is limited by the stability of the proteins bound on its crystal surface. Antibodies and antibody-binding proteins require cold storage and lose binding activity with time. Still another drawback is that some of the proteins can be attached to the surface in an orientation that obscures binding sites for the compound of interest. Additionally, the procedure for immobilizing the proteins on the surface exposes them to chemicals which can lower binding activity by affecting functional groups at the binding sites. Furthermore, the procedure can require additional modifications for each specific protein system. Yet another problem is the high degree of nonspecific adsorption on the surface: many molecules in solution will bind to the surface by means of weak electrostatic and hydrogen bonds. In a mass biosensor, this nonspecific binding affects the measurement, limiting the sensitivity of the instrument and its analytical and clinical usefulness.
What is needed is a quick and convenient procedure for customizing a sensor to bind chemicals of interest for diverse applications. Additionally needed is a sensor with minimal nonspecific binding so that it can be used in detecting trace quantities of chemicals of interest.
In accordance with the present invention, a measuring device includes a measurement surface, a ligand-binding layer on the surface, and a ligand-bearing layer bound to the ligand-binding layer. The ligand-bearing layer is selected for its binding affinity to a chemical to be measured. For example, a layer of a selected biotinylated antibody can be bound to an avidin layer, in turn bound to the piezoelectric crystal substrate of a mass biosensor.
Preparation of the measuring device involves chemically modifying the binding properties of the measurement surface by utilizing a ligand-conjugated compound having binding affinity for a chemical of interest and also utilizing a substance having reciprocal ligand-binding sites. The preparation steps comprise attaching the ligand-binding substance as a layer to the surface, binding the ligand-bearing compound as a layer to the ligand-binding substance, and washing the resultant structure with a blocking agent which covers free active sites in order to reduce non-specific adsorption. The resulting composite surface can be used for selective binding of the chemical of interest. As an example, avidin can be coupled to a silica substrate, a biotinylated antibody can be attached to the avidin, and biotin can be added to block unoccupied active sites. This composite surface will bind tightly to antigen with minimal nonspecific adsorption.
As indicated, avidin is a favored material for the ligand-binding layer and biotinylated antibodies are appropriate for the ligand-bearing layer. A biotinylated antibody is composed of an antibody bound to biotin directly or via a spacer molecule. Biotin has one avidin-binding site per molecule and avidin has four biotin-binding sites per molecule. Avidin and biotin bind tightly to one another through their reciprocal binding sites, even when each is in turn conjugated to other molecules, as long as the high affinity binding sites are not blocked.
The present invention provides a device which, when immersed in a liquid, binds selected chemicals. The surface of the device has overlaying layers composed of ligand-binding substance and ligand-conjugated compound, the latter having strong binding affinity toward the chemicals of interest. The ligand-binding substance can be bound to the substrate using a coupling agent. Nonspecific binding can be controlled by pretreatment with blocking agents, buffers, or other regimen. Blocking agents are chemicals that bind to the device and sterically inhibit the nonspecific binding from weak bond formation. Buffers of appropriate pH can inhibit nonspecific binding by neutralizing charged sites on the surface.
A mass biosensor provided by the present invention includes an electric signal generator, an electro-acoustic input transducer, a piezoelectric crystal waveguide, an electro-acoustic output transducer, and means for measuring phase changes between the electric signals from the generator and output transducer. In this case, the waveguide is immersed in a liquid, a signal is applied, and the phase or frequency of the output signal from the waveguide is monitored. Mass changes on the surface of the waveguide affect the output signal and the mass or concentration of the analyte can be calculated from these data.
The present invention provides for preparation of the measurement surface in two phases to obviate problems of excessive preparation time and product instability. In accordance with the preferred embodiment, the first phase of preparation involves attaching avidin to the sensor surface. This phase, which is time consuming, can be accomplished efficiently by a manufacturer of the device. Avidin retains its binding activity at room temperature so that the avidin-coated device has a relatively long shelf life and, for manufacturing purposes, can be packaged and distributed without cold storage at considerable cost saving. A further advantage to the manufacturer is that avidin, which is a constituent of egg whites, can be abundantly supplied at low cost. Still another advantage for applications requiring sterile conditions is that the avidin-modified sensor can be sterilized since avidin withstands temperatures up to 120xc2x0 C.
The second phase involves attaching a biotinylated compound to the avidin and blocking weak binding sites on the surface. This phase can be accomplished with ease by the user in about 30 minutes. Although biotinylated antibody is less stable than avidin, this component need not be added to the surface until just prior to use. Another advantage to the user is that the second phase of preparation requires no expensive apparatus or chemicals. Further, this phase of the procedure is accomplished at room temperature under physiological conditions so that the binding activity of the biotinylated compounds is not compromised by harsh chemicals or high temperatures. Of great convenience to the user is the fact that biotinylated compounds are available commercially or can be prepared following published procedures. Thus, the present invention significantly enhances the convenience with which mass biosensors can be customized to assess the amounts of selected chemicals.
An important feature of the present invention is that it can reduce nonspecific binding. Free, active binding regions on the silica surface are sterically blocked by bound avidin. Free, unbound sites on avidin are blocked in the final wash step with biotin. Since biotin is small and of low molecular weight, it has access to unbound binding sites that may otherwise be inaccessible to larger blocking agents because of steric hindrance.
Yet another feature of the invention is the multiplication of binding sites. Since a single molecule of avidin has four biotin binding sites, when a silica surface is overlaid with avidin, the number of potential binding sites for biotinylated derivatives is fourfold the number of binding sites for avidin. When using biotinylated antibodies for the next layer, each of which contains two antigen binding sites, potential binding sites for the antigenic species are amplified again. These and other features and advantages of the present invention will be apparent from the description below with reference to the following drawings.
FIG. 1 is a schematic view of a sensor utilizing a surface transverse wave device with a chemically modified top surface.
FIG. 2 is a schematic illustration of the process for immobilizing avidin to a substrate.
FIG. 3 is a schematic illustration of the process for attaching a biotinylated antibody to an avidin-coated substrate.